Microbes and Infection 10 (2008) 334e341www.elsevier.com/locate/micinf

Original article

Decreased inflammatory response in Toll-like receptor 2 knockoutmice is associated with exacerbated Pneumocystis pneumonia

Shao-Hung Wang a, Chen Zhang b, Mark E. Lasbury b, Chung-Ping Liao b,Pamela J. Durant b, Dennis Tschang b, Chao-Hung Lee b,*

a Department of Medical Research, E-DA Hospital, I-Shou University, Kaohsiung 824, Taiwan, ROCb Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Received 9 November 2007; accepted 17 December 2007

Available online 28 December 2007

Abstract

Pneumocystis pneumonia (PcP) is marked by substantial inflammatory damage to the lung. We have found that Toll-like receptor 2 (TLR2)mediates macrophage inflammatory responses to Pneumocystis and hypothesized that TLR2 deficiency would lead to less severe inflammationand milder lung injury during PcP. Histopathology examination showed that TLR2�/� mice with PcP indeed exhibited milder pulmonaryinflammation. TLR2�/� mouse lungs contained less TNF-a and displayed lower levels of NF-kB activation during PcP. However, TLR2�/�mice with PcP displayed increased severity in symptoms and organism burden. The increased organism burden is likely due to defects in pro-tective mechanisms in TLR2�/� mice. mRNA levels of the inducible nitric oxide synthase and NADPH oxidase p47phox, as well as nitric oxidelevels in the lungs, were decreased in TLR2�/� PcP mice. Taken together, this study shows that TLR2-mediated inflammatory responses con-tribute to a certain degree to the clearance of Pneumocystis organism in mice.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Pneumocystis; Toll-like receptor 2; Inflammation

1. Introduction

Pneumocystis pneumonia (PcP) is the most commonopportunistic disease in immunocompromised hosts, such aspatients with AIDS [1]. Pneumocystis organisms that infecthumans are now referred to as Pneumocystis jirovecii [2], andthose that infect rats and mice are called Pneumocystis cariniiand Pneumocystis murina, respectively [2,3]. Although therehas been a significant decline in incidence of PcP due to theuse of highly active antiretroviral therapy (HAART) [4], PcPremains a significant cause of morbidity and mortality in humanimmunodeficiency virus (HIV)-infected patients and a life-threatening disease in other immunocompromised patients.

* Corresponding author. Department of Pathology and Laboratory Medicine,

Indiana University School of Medicine, 1120 South Drive, Fesler Hall Rm.

419, Indianapolis, IN 46202, USA. Tel.: þ1 317 274 2596; fax: þ1 317 278

0643.

E-mail address: chlee@iupui.edu (C.-H. Lee).

1286-4579/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.micinf.2007.12.014

Alveolar macrophages play an important role in the clear-ance of organism from the lungs, and the phagocytosis is medi-ated by interactions between the pathogen and various types ofreceptors. Several surface molecules on alveolar macrophageshave been shown to interact with Pneumocystis, includingFc-gamma receptor [5], complement receptor [6], scavengerreceptor [7], mannose receptor [8], and dectin-1 [9]. TLR2 isa pattern recognition receptor recognizing a large variety of li-gands such as peptidoglycan, lipoprotein, lipopeptide, andzymosan [10]. Activated TLR2 transduces signals througha myeloid differentiation factor 88 (MyD88)-dependent path-way, inducing nuclear translocation of the transcription factorNF-kB [10]. NF-kB activation is crucial for the production ofproinflammatory cytokines and chemokines that initiate hostinflammatory response. We have recently demonstrated thatTLR2 mediates the alveolar macrophage inflammatory re-sponse to Pneumocystis in vitro [11]. The role of TLR2response against Pneumocystis in the host remains undefined.

335S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

In this study, we show that TLR2 plays a major role in theinflammatory response to Pneumocystis infection in vivo.

2. Materials and methods

2.1. Mouse model of PcP

TLR2-deficient mice (TLR2�/�, C57BL/6 background)were purchased from the Jackson Laboratory (Bar Harbor,ME). Wild type C57BL/6 mice were obtained from Harlan(Indianapolis, IN). All mice were female, 6e8 weeks of age,and 18e20 g in weight at arrival. Animal studies wereapproved by the Indiana University Animal Care and UseCommittee. Animals were housed in the Indiana UniversityLaboratory Animal Resource Center, an American Associationfor Accreditation of Laboratory Animal Care (AAALAC) ap-proved facility. All mice were immunosuppressed by weeklyintraperitoneal injection of 0.3 mg anti-CD4 mAb (cloneGK1.5, Harlan, Indianapolis, IN). Flow cytometry analysis in-dicated that 0.3 mg of the antibody achieved 95% depletion ofCD4þ cells after a single injection. This result was consistentwith that of Zheng et al. [12]. Immunosuppressed mice weretranstracheally instilled with 2 � 106 P. murina organisms.Immunosuppressed, non-infected mice were used as controls.Tetracycline was added to the drinking water (9.2 g/L) toprevent bacterial infections. Mice were sacrificed at 8 weeksafter infection or when they displayed four of the followingcriteria: weight loss below 65% of arrival weight, ability topalpate bony structures such as the spine, dark appearanceof the eyes, dried blood around the nares, extremely inactiveand labored breathing.

2.2. Histopathology examination

Mice were anesthetized and sacrificed by cardiac exanguina-tion. The lungs were fixed and embedded in paraffin. Histologicsections were stained with H&E and evaluated for the followingfeatures: perivascular and peribronchiolar mononuclear infiltra-tion, neutrophil infiltration, foamy exudates, fibrosis, and ef-facement of alveolar structure. Pulmonary inflammation wasgraded blindly by two experienced microscopists usinga semi-quantitative scale described by Rudmann et al. [13].Scores of 1e5 were based upon grading of the entire lung tissuepresent on the sections. To determine the organism burden, lungsections were stained with the GMS silver staining kit (SigmaChemical Co., St. Louis, MO) and examined under lightmicroscope.

2.3. Single cell preparation from mouse lungs

Mice were anesthetized, and pulmonary vasculature was per-fused via right ventricle with 10 ml cold Dulbecco’s phosphatebuffered saline (D-PBS). Lungs were inflated with 2 ml ofDispase II (Sigma) at 5 mg/ml with an intratracheal catheter.Trachea was ligated with suture silk to prevent leakage of Dis-pase II. Lungs were incubated in D-PBS for 45 min at 37 �C andthen minced well with scissors and scalpels. Minced lungs were

incubated in 5 ml D-PBS containing 1 mg/ml of Collagenase/Dispase (Roche, Indianapolis, IN) and 0.08 mg/ml of DNase IType II (Sigma) for 10 min at 37 �C with shaking. Digestedcells were filtered through a 70 mm nylon mesh (BD Biosci-ences, Franklin Lakes, NJ), followed by a 40 mm nylon mesh(BD Biosciences).

2.4. Analysis of inflammatory cells in singlecell suspensions by flow cytometry

Approximately 1 million cells were stained with fluores-cence labeled antibodies for monocytes/macrophages (Phyco-erythrin-labeled anti-Mac3, R&D systems, Minneapolis, MN),neutrophils (Fluorescein isothiocyanate-labeled anti-Ly-6G,eBioscience, San Diego, CA), and CD8þ lymphocytes (Fluo-rescein isothiocyanate-labeled anti-CD8, eBioscience). Thestained cells were analyzed by a BD FACScan flow cytometer(BD Biosciences). Percentages of monocytes/macrophages,neutrophils, and CD8þ lymphocytes in total lung cells weredetermined.

2.5. Determination of arterial blood oxygen tension

After gentle heating with a lamp, the mice were restrainedand heparin was swabbed onto the skin to prevent clotting.The ventral artery of the tail was nicked by plunging a scalpelblade diagonally into the artery. Approximately 100 ml ofblood was collected in a heparinized syringe and immediatelyplaced on ice. The samples were analyzed on a clinical bloodgas machine within 60 min of collection.

2.6. RNA isolation and real-time RT-PCR assay

Total RNA was isolated from lung tissue using the TRIzolreagent according to manufacturer’s instructions (Invitrogen,Carlsbad, CA). cDNA was synthesized using the iScriptcDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time RT-PCR for TNF-a, MIP-2, iNOS, and p47phox were performedusing the Assays-on-Demand� gene expression kits (AppliedBiosystems, Foster City, CA) as previously described [11].The mRNA level of RPS8 was used as an internal controlsince mRNA levels transcribed from this gene are not alteredby Pneumocystis infection [11].

2.7. Quantification of P. murina organismburden by real-time RT-PCR

Organism burdens in the lungs were estimated based on therRNA levels of P. murina mitochondrial large subunit ribo-somal RNA gene (GenBank accession no. AF257179) usingthe method described by Zheng et al. [12]. A portion of theP. murina mitochondrial large subunit ribosomal RNA genewas cloned, and Pc rRNA was produced by in vitro transcrip-tion. A standard curve was generated with the known copynumbers of P. murina rRNA. Total RNA from infected lungswas purified and real-time RT-PCR for Pc rRNA wasperformed using the TaqMan� One-Step RT-PCR Master

336 S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

Mix Reagent Kit (Applied Biosystems). Ct values of thesamples were converted to rRNA copy number using thestandard curve.

2.8. Quantification of TNF-a in bronchoalveolarlavage fluids (BALF) by ELISA

Lungs were lavaged with 1 ml sterile saline through an in-tratracheal catheter as described previously [11]. BALF wascentrifuged at 10,000 � g for 5 min to remove cellular debris.TNF-a concentrations in BALF were determined by sandwichELISA as described previously [11].

2.9. Determination of NF-kB activation byelectrophoretic mobility shift assay (EMSA)

A biotin-labeled consensus double-stranded NF-kB oligo-nucleotide probe (50-AGTTGAGGGGACTTTCCCAGGC-30)was used for EMSA. Nuclear extract was obtained fromlung tissue homogenates using the nuclear extraction kit(Active Motif, Carlsbad, CA). Two micrograms of nuclearprotein was used to detect NF-kB activation using the Light-Shift Chemiluminescent EMSA Kit (Pierce, Rockford, IL).Cold competition was carried out using specific unlabeledprobe. Unlabeled oligonucleotide that does not bind NF-kBwas used as non-specific competitor.

2.10. Quantification of nitrate and nitrite in BALF

Since nitric oxide is quickly oxidized to nitrite and nitrate,nitrite levels were determined. Briefly, freshly obtained BALFwas incubated with cadmium granules to reduce nitrate to ni-trite, followed by adding Griess reagent of the QuantiChrom�Nitric Oxide Assay Kit (BioAssay Systems, Hayward, CA),and absorbance at 540 nm was measured using a microplatereader (Molecular Dynamics, Sunnyvale, CA). Total nitriteconcentration was calculated from the standard curve formedby serial dilution of nitrite standards.

2.11. Statistics

Data were presented as mean � standard deviation (SD) ofthe indicated number of experiments. Differences betweengroups were determined using the two-tail Student’s t-testand were considered statistically significant if p was <0.05.

3. Results

3.1. Decreased inflammation in the lungs ofTLR2�/� mice with PcP

To examine the role of TLR2 in the inflammatory responseto Pneumocystis infection, wild type and TLR2�/� mice weresacrificed eight weeks after infection with P. murina.Inflammation in the lungs was evaluated by histopathology ex-amination. As shown in Fig. 1, lungs of wild type mice withPcP displayed severe inflammation, characterized by increased

numbers of monocytes, lymphocytes, neutrophils, and eosino-phils and destruction of alveolar parenchyma and smallairways. In contrast, there were fewer mononuclear cells andless neutrophil infiltration in the lungs of TLR2�/� micewith PcP. Although there was more foamy exudate in thealveoli, the alveolar structure was better preserved in the lungsof TLR2�/� mice with PcP (Fig. 1). The pulmonary inflam-mation score of TLR2�/� PcP mice was significantly lowerthan that of the wild type mice with PcP (3.6 � 0.4 vs.4.5 � 0.7, p ¼ 0.012). Flow cytometry analysis of single cellpreparations showed that the percentage of neutrophils in totallung cells was decreased by 86% (0.71 � 0.38% vs. 5.13 �0.93%, p ¼ 0.007), and that of monocyte/macrophage wasdecreased by 67% (3.99 � 1.02% vs. 12.10 � 1.15%, p ¼0.02) in the lungs of TLR2�/� mice with PcP compared tothose of wild type mice with PcP. The number of CD8þ lym-phocytes in the lungs of TLR2�/� mice with PcP was notsignificantly different from that of wild type mice with PcP(0.90 � 0.37% vs. 1.14 � 0.46%, p ¼ 0.165).

3.2. Decreased production of TNF-a inTLR2�/� mice with PcP

To investigate whether the decreased inflammation in thelungs of TLR2�/� mice with PcP was due to decreasedTNF-a production, TNF-a mRNA expression levels of lungtissue and TNF-a protein levels in the BALF were determined.As shown in Fig. 2, TNF-a mRNA abundance (Fig. 2a) andTNF-a production (Fig. 2b) were both increased in wild typemice and TLR2�/�mice during P. murina infection. However,compared to wild type mice with PcP, TLR2�/�mice with PcPshowed significantly lower levels of TNF-a mRNA expression(39.5 � 16.8 fold increase vs. 90.5 � 18.7 fold increase,p ¼ 0.009) and TNF-a production (90.0 � 12.2 pg/ml vs. 172.8� 18.4 pg/ml, p ¼ 0.043). This result suggests that TLR2 ispartially involved in TNF-a production in response to Pneumo-cystis. Similar pattern was found in the mRNA expression levelof another pro-inflammatory cytokine, MIP-2. TLR2�/� micewith PcP showed significantly lower levels of MIP-2 mRNAexpression (41.8 � 11.3 fold increase vs. 91.7 � 31.6 foldincrease, p ¼ 0.02).

3.3. Decreased NF-kB activation in the lungsof TLR2�/� mice with PcP

Since the expression of TNF-a is regulated by NF-kB [10],the role of TLR2 in the activation of NF-kB during PcP wasexamined. Lung homogenates were used to isolate nuclearextracts for determination of NF-kB activation by EMSA.As shown in Fig. 3, P. murina-induced NF-kB activationwas seen in both wild type mice and TLR2�/� mice, butTLR2�/� mice with PcP showed a markedly reduced levelof NF-kB activation in the lungs. Densitometry analysis ofthe autoradiograph shown in Fig. 3 indicated that Pneumocys-tis infection induced 1.3 fold and 3.48 fold increases of NF-kBactivation in TLR2�/� mice and wild type mice, respectively.These data are consistent with the less robust TNF-a response

Fig. 1. Decreased inflammation in the lungs of TLR2�/� mice with PcP. Immunosuppressed wild-type and TLR2�/� mice were infected with P. murina for eight

weeks. Lungs were fixed, embedded, and sectioned. Lung sections were stained with H&E, and examined with light microscopy at magnifications of 100� and

400�. Data presented are representative for 6e8 mice per group. Similar results were obtained in three independent experiments. Scale bar ¼ 100 mm.

337S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

in TLR2�/� PcP mice as compared to wild type PcP miceshown in Fig. 2, suggesting that TLR2 deficiency results inless NF-kB activation, TNF-a production, and inflammationin the lungs during P. murina infection.

3.4. More severe PcP in TLR2�/� mice

To investigate the progression of PcP, wild type andTLR2�/� mice with PcP were examined for several parame-ters of disease. As shown in Fig. 4, both wild-type andTLR2�/� mice started to lose weight 6 weeks after initiationof infection, but the loss was greater in TLR2�/� mice than inwild type mice at 7 and 8 weeks of infection. TLR2�/� micewith PcP displayed more severe symptoms of disease includ-ing reduced grooming, dyspnea, and hypokinesis after 8 weeks

of infection. The survival rates of TLR2�/� mice and wildtype mice at 8 weeks of infection were 78.6% and 96.4%,respectively (n ¼ 28). Arterial blood oxygen tension (PaO2)of TLR2�/� mice with PcP was significantly lower thanthat of wild type mice with PcP (67.8 � 0.5 mmHg vs. 80.1 �3.5 mmHg, p ¼ 0.03, n ¼ 8).

3.5. Increased organism burden in the lungs ofTLR2�/� mice with PcP

Silver staining of the lung tissue sections from TLR2�/�mice with PcP showed a significantly higher level of P. murinacyst burden than those from wild type mice with PcP (Fig. 5a).The increased organism burden was confirmed by real-timeRT-PCR for Pneumocystis rRNA with an average of

Fig. 2. Decreased production of proinflammatory cytokine TNF-a in TLR2�/�mice with PcP. (a) Total RNA was isolated from lung cells of uninfected mice

and those that had been infected for eight weeks. TNF-a mRNA expression

levels were determined by real-time RT-PCR. The mRNA of the ribosomal

protein S8 (RPS8) gene was co-amplified with that of TNF-a to serve as the

internal PCR control in each assay. Fold increase in TNF-a mRNA levels in

the lungs is relative to that of the lungs from uninfected wild-type control

mice, which was set to a value of one. (b) BALF of the same infected or un-

infected mice was analyzed for TNF-a protein level (pg/ml) by ELISA. Bars

represent means � SD for 6e8 mice per group. Similar results were obtained

in three independent experiments.

Fig. 3. Decreased NF-kB activation in the lungs of TLR2�/� mice with PcP.

Nuclear protein was isolated from lung cells of uninfected mice and those that

had been infected for eight weeks. NF-kB activation level in the nuclear

extract was determined by EMSA. The blank lane is the control reaction

mixture containing biotin-labeled DNA probe without adding nuclear extract.

Data presented are representative for 6e8 mice per group. Similar results were

obtained in three independent experiments.

Fig. 4. Body weight change in Pneumocystis-infected wild type and TLR2�/�mice. The mice were weighed every week, and changes in body weight (g)

were calculated. Bars represent means � SD for 6e8 mice per group. Similar

results were obtained in three independent experiments.

338 S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

6.71 � 108 copies in the lungs of TLR2�/� mice and 2.99 �108 copies in those of wild type mice with PcP ( p ¼ 0.04)(Fig. 5b). These data suggest that TLR2 knockout leads to a re-duced clearance of P. murina organisms.

3.6. Impaired inducible nitric oxide synthase(iNOS) induction and nitric oxide production inTLR2�/� mice with PcP

To explore a possible reason why TLR2�/� mice were lessable to clear P. murina organisms, iNOS mRNA levels in thelungs and nitric oxide levels in the BALF were determined. Asshown in Fig. 6a, iNOS mRNA expression was significantlyincreased in wild type mice and TLR2�/� mice during P.murina infection. However, compared to wild type mice withPcP, TLR2�/� mice with PcP showed significantly lowerlevels of iNOS mRNA expression (949.1 � 193.8 fold in-crease vs. 1818.2 � 216.0 fold increase, p ¼ 0.022). Determi-nation of nitric oxide levels in BALF showed that TLR2�/�mice with PcP contained significantly less nitric oxide thanwild type mice with PcP (20.1 � 2.6 mM vs. 27.1 � 4.5 mM,p ¼ 0.012) (Fig. 6b).

3.7. Impaired NADPH oxidase induction inTLR2�/� mice with PcP

NADPH oxidase is the key enzyme in the production ofreactive oxygen species (ROS). To investigate the effect ofTLR2 knockout on ROS production during PcP, the mRNAlevel of NADPH oxidase p47phox subunit in the lungs wasdetermined. As shown in Fig. 6c, p47phox mRNA abundancewas significantly increased in wild type mice and TLR2�/�mice during P. murina infection. However, compared to wildtype mice with PcP, TLR2�/� mice with PcP showed a signif-icantly smaller increase in p47phox mRNA (17.7 � 6.5 foldincrease vs. 59.6 � 5.7 fold increase, p ¼ 0.03).

4. Discussion

As a pattern recognition receptor that recognizes fungal cellwall components, TLR2 was shown in our previous study to

Fig. 5. Increased organism burden in TLR2�/�mice with PcP. Immunosuppressed wild-type and TLR2�/�mice were infected with P. murina for eight weeks. (a)

Lungs were fixed, embedded, and sectioned. Lung sections were stained with the GMS silver staining kit, and examined with light microscopy at magnifications of

100� and 200�. Results shown are representative of 6e8 mice per group. (b) Total RNA was isolated from the lungs and subjected to real-time RT-PCR for Pc

rRNA. Pc rRNA copy numbers in the lungs of wild type and TLR2�/� mice with PcP were calculated from a standard curve generated with known copy numbers

of Pc rRNA. Bars represent means � SD for 6 mice per group.

339S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

mediate alveolar macrophage inflammatory response to P.murina in vitro [11], but the role of TLR2 in the host responseduring Pneumocystis pneumonia had not been determined. Inthis study, we used TLR2�/� mice to investigate the rolesof TLR2 in the host response against Pneumocystis infectionand found that TLR2 knockout mice with PcP had less severepulmonary inflammation than wild type mice with PcP(Fig. 1). We also found that TLR2�/� mice with PcP dis-played less TNF-a in BALF (Fig. 2) and lower level of NF-kB activation than the wild type mice with PcP (Fig. 3). SinceNF-kB regulates the expression of TNF-a [10], a reduction inNF-kB activation would certainly result in a decreased produc-tion of TNF-a. TLR2 is one of many receptors that can triggerNF-kB activation. It is conceivable that TLR2 knockout leadsto diminished NF-kB activation, reduced TNF-a production,and subsequently less pulmonary inflammation.

Although the levels of NF-kB activation and TNF-a produc-tion were significantly lower in TLR2�/� mice with PcP thanin wild type mice with PcP, they were still significantly higherthan those in uninfected control mice. TLR2�/� mice withPcP still showed some degree of pulmonary inflammation(Fig. 1), suggesting that TLR2 is not the only receptor that me-diates inflammatory response to Pneumocystis in vivo. This isconsistent with previous studies showing that other receptors,such as dectin-1 and mannose receptors, also play importantroles in the host response to Pneumocystis infection [9,14].Other studies have shown that TLR2 collaborates with dec-tin-1 and mannose receptors to mediate inflammatory responseto Pneumocystis [15,16]. A recent review by Dennehy andBrown described that dectin-1 is critical in the control of fun-gal infection since it can mediate its own signaling, as well assynergize with TLR to initiate responses to fungal pathogens

Fig. 6. Impaired iNOS induction, nitric oxide production, and NADPH oxidase

induction in TLR2�/� mice with PcP. (a) Total RNA was isolated from lung

cells of uninfected mice and those that had been infected for eight weeks, and

iNOS mRNA expression levels were determined by real-time RT-PCR. The

mRNA of the RPS8 gene was co-amplified with that of iNOS to serve as

the internal PCR control in each assay. Fold increase in iNOS mRNA levels

in the lungs is relative to that of the lungs from uninfected wild-type control

mice, which was set to a value of one. (b) Nitrate and nitrite levels (mM) in

BALF were determined by using the Griess reagent of the QuantiChrom� Ni-

tric Oxide Assay Kit. (c) Total RNA was isolated from cells of infected and

uninfected lungs, and p47phox mRNA levels were determined by real-time

RT-PCR. Fold increase in p47phox mRNA levels in the lungs is relative to

that of the lungs from uninfected wild-type control mice, which was set to

a value of one. Bars represent means � SD for 6e8 mice per group. Similar

results were obtained in three independent experiments.

340 S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

[17]. Study by Ding et al. showed that there was a reducedlevel of MIP-2 in bronchoalveolar lavage fluid of TLR4mutant mice with PcP, suggesting that TLR4 also mediatesthe inflammatory response to Pneumocystis [18].

Host defense against infection is critically dependent uponrecruitment of immune cells into infected tissue. These cellsmay include lymphocytes, neutrophils, monocytes and macro-phages, and serve as the effecter cells for the clearance ofmicroorganisms. However, over-exuberant inflammation maycause tissue damage. In fact, inflammation is considered themajor cause of lung damage in PcP. Wright et al. have foundthat immune-mediated inflammation directly impairs pulmo-nary function during PcP [19]. A recent study showed that

sensitized CD8þT cells fail to clear organisms but acceleratethe onset of lung injury during PcP [20]. In this study, wefound that the numbers of neutrophil and monocyte/macro-phage were decreased in TLR2�/� mice with PcP and thenumber of CD8þT cells in the lungs of TLR2�/� micewith PcP was found to be similar to that of wild type micewith PcP. These results suggest that neutrophil and mono-cyte/macrophage play a role in TLR2-mediated inflammatoryresponse during PcP. Whether this inflammation leads to lungdamage remains to be investigated since it has been shown thatneutrophils do not contribute to lung damage during PcP [21].The observation that TLR2�/� mice developed more severePcP symptoms with increased organism burden as comparedto wild type mice (Figs. 3 and 4) indicates that TLR2-mediatedresponse contributes to the elimination of Pneumocystisorganisms.

Clearance of the organisms by the host is mediated bydirect phagocytosis, and the production of toxic antimicrobialmetabolites such as nitric oxide and ROS by macrophages.Nitric oxide has been shown to be an important antimicrobialsubstance. Pneumocystis organisms can stimulate alveolarmacrophages to produce nitric oxide [22], and nitric oxide istoxic to Pneumocystis organisms [23]. iNOS is the key enzymein the production of nitric oxide. The promoter region of themouse iNOS gene contains several binding sites for transcrip-tion factors including NF-kB [24]. Therefore, it is possible thata reduction in NF-kB leads to a decreased production of nitricoxide. This is indeed the case in the TLR2�/� mice with PcP,as iNOS mRNA levels in lung homogenates and nitric oxideconcentration in BALF from these mice were significantly de-creased as compared to wild type mice with PcP (Fig. 6a,b).This may account for the defect in the clearance of the organ-ism in TLR2�/� mice. However, the roles of nitric oxide andiNOS in the clearance of Pneumocystis organisms require to bemore carefully investigated as both molecules have beenshown to be inadequate in the control of Pneumocystis infec-tion [25].

ROS, such as super oxide and hydrogen peroxide, are alsoimportant antimicrobial intermediates. Pneumocystis can in-duce ROS production in alveolar macrophages [26]. A recentstudy shows that ROS are important in the protection againstPneumocystis infection because defective production of ROScorrelated with increased susceptibility to Pneumocystis infec-tion in dectin-1 knockout mice [27]. Superoxide production isregulated by the multisubunit enzyme NADPH oxidase. In ourstudy, the mRNA level of the p47phox component of NADPHoxidase was found to be much lower in TLR2�/� mice withPcP (Fig. 6c). The impaired production of ROS in TLR2�/�mice may be another reason for the decreased clearance ofPneumocystis organisms.

Since TLR2�/� mice with PcP exhibited more severesymptoms despite decreased inflammation in the lungs, theremust be factors other than inflammation that cause theimpaired lung function. The increased organism burden maydirectly impair lung function by disrupting oxygen diffusioninside the alveoli. Previous studies suggest that Pneumocystisorganisms require attachment or close apposition to the

341S.-H. Wang et al. / Microbes and Infection 10 (2008) 334e341

alveolar epithelium in order to proliferate [28] An ultrastruc-tural study shows that cysts and trophozoites are held togetherby slender membranotubular extensions to form the foamy al-veolar cast on the surface of the pulmonary epithelium, whichmay impair the gas exchange function of the alveoli [29].

In this study, wild type mice with PcP had more severe in-flammation, but had lower organism burden, less body weightloss, and most importantly a higher survival rate than TLR2�/�mice with PcP. This result suggests that inflammation is one ofthe host defense mechanisms against Pneumocystis infectionand that anti-inflammatory therapy for PcP needs to be usedwith caution. A complete inhibition of inflammation mayhave adverse effects. There have been reports of clinical ben-efit from the use of adjunctive corticosteroids in addition to thespecific anti-Pneumocystis therapy. Some of the benefits arerelated to the decreased hypersensitive reactions to antibiotictherapy. However, a small, but significant, increased rate of in-fection in steroid-treated patients is noted [30]. The anti-in-flammatory therapy for PcP is beneficial only in conjunctionwith effective anti-microbial treatments [30].

Acknowledgement

The study was supported by grants from the NationalInstitutes of Health (RO1 HL65170 and RO1 AI062259).

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